Temperature measurement enzyme reaction detection

A FI calorimetric biosensor was developed for the determination of dichlorvos (Zheng et al., 2006). The enzyme chicken liver esterase was used as the biorecognition element and acetyl-l-naphthol as the substrate. This enzyme was immobilized on an ion exchange resin, which was then packed in the enzyme reaction cell. The reference cell was filled with the same batch of the resin, but with a completely inactivated enzyme. As a result, the enzymatic reaction occurred in the enzyme reaction cell, but not in the reference cell and there was a temperature difference at the outlets of the two cells. The detection was based on the inhibition of the enzyme by the analyte, measuring the difference of the signal obtained with and without inhibition. 

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 340-352 measurement, 63, 365 cryosolvent [catalytic effect, 63, 344-346 choice, 63, 341-343 dielectric constant, 63, 354 electrolyte solubility, 63, 355, 356 enzyme stability, 63, 344 pH measurements, 63, 357, 358 preparation, 63, 358-361 viscosity effects, 63, 358] intermediate detection, 63, 349, 350 mixing techniques, 63, 361, 362 rapid reaction techniques, 63, 367-369 temperature control, 63, 363-367 temperature effect on catalysis, 63, 348, 349 temperature effect on enzyme structure, 63, 348. 

To purify a protein, it is essential to have a way of detecting and quantifying that protein in the presence of many other proteins at each stage of the procedure. Often, purification must proceed in the absence of any information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are enzymes, the amount in a given solution or tissue extract can be measured, or assayed, in terms of the catalytic effect the enzyme produces—that is, the increase in the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose one must know (1) the overall equation of the reaction catalyzed, (2) an analytical procedure for determining the disappearance of the substrate or the appearance of a reaction product, (3) whether the enzyme requires cofactors such as metal ions or coenzymes, (4) the dependence of the enzyme activity on substrate concentration, (5) the optimum pH, and (6) a temperature zone in which the enzyme is stable and has high activity. Enzymes are usually assayed at their optimum pH and at some convenient temperature within the range... 

After optimizing the assay conditions, including ionic strength, pH, temperature, activator (Ca ) concentration, and polymer concentration, a calibration curve was developed, which allows the lipid substrate concentration to be determined from the fluorescence intensity. The calibration curve allows the enzyme catalysis kinetics parameters (e.g.. Km and Vmax) to be measured. This PLC turn-off assay is effectively inhibited by known inhibitors (F and EDTA), which demonstrates that the sensor relies on the specific catalysis reaction by PLC. It has been demonstrated to be a sensitive (detection limit 0.5nM enzyme concentration), fast (<5 min), and selective (good specificity over phospholipase A and D, and other nonspecific proteins) PLC assay, which can be carried out at very low initial substrate concentration (in the range of micromolar to nanomolar). 

Table IV summarizes the results of exonuclease activity measurements (expressed as specific activity (U/mg)) conducted at optimal temperature in the absence of dNTPs. For certain DNA polymerases, assays were carried out in both the common assay buffer and in each enzyme s optimal PCR reaction buffer. Inclusion of ammonium sulfate in PCR buffers was found to significantly increase the exonuclease activity of archaeal DNA polymerases (data not shown). In assays employing uniformly labeled double-stranded DNA substrates, release of acid-soluble nucleotides can be attributed to the 3 ->-5 -exonuclease activity associated with archaeal Family B DNA polymerases, UlTma, and KF, as these enzymes are reportedly devoid of detectable 5 ->-3 -exonuclease activity (reviewed ). Release of acid-soluble nucleotides by nonproofreading DNA polymerases, such as Taq, can be attributed to 5 3 exonuclease activity, and solubilized radioactivity increases in the presence of added dNTPs (data not shown). No detectable exonuclease activity was observed for the exo DNA polymerase, whereas the 9°N mutant exhibited l-3% of the exonuclease activity of other archaeal DNA polymerases, consistent with the reduced exonuclease activity noted by the enzyme s manufacturer.

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